HIV Infection and AIDS
Human Immunodeficiency Virus (HIV), the etiological agent for AIDS (Acquired Immune Deficiency Syndrome), is a member of the lentiviruses, a subfamily of retroviruses. Many retroviruses are well-known carcinogens. HIV per se is not known to cause cancer in humans or other animals, but it does present a formidable challenge to the host. HIV integrates its genetic information into the genome of the host. The viral genome contains many regulatory elements which allow the virus to control its rate of replication in both resting and dividing cells. Most importantly, HIV infects and invades cells of the immune system; it destroys the body's immune system and renders the patient susceptible to opportunistic infections and neoplasms. The immune defect appears to be progressive and irreversible, with a high mortality rate that approaches 100% over several years.
HIV is transmitted by parenteral inoculation and/or intimate sexual contact. It is estimated that about 2 million people in the United States are currently infected with HIV, and 5 to 10 million people are infected worldwide. Recent projections indicate that a majority of those now infected will develop AIDS within a seven year follow-up period. In 1989 alone, over 130,000 cases of AIDS were reported domestically, and more than half of these patients have died. It is estimated that an additional 200,000 cases will be diagnosed in the United States by the end of 1990. Reports to the World Health Organization suggest that at least a million new cases of AIDS can be expected within the next five years worldwide. It is apparent that AIDS is an unprecedented threat to U.S. as well as global health. The search for effective therapies to treat AIDS is of paramount importance.
HIV-1 is trophic and cytopathic for T4 lymphocytes, cells of the immune system which express the cell surface differentiation antigen CD4 (also known as OKT4, T4 and leu3). The viral tropism is due to the interactions between the viral envelope glycoprotein, gp120, and the cell-surface CD4 molecules (Dalgleish, A. G. et al., Nature 312: 763-767 (1984). These interactions not only mediate the infection of susceptible cells by HIV but are also responsible for the virus-induced fusion of infected and uninfected T cells. This cell fusion results in the formation of giant multinucleated syncytia, cell death, and progressive depletion of CD4 cells in AIDS patients. These events result in HIV-induced immunosuppression and its subsequent sequelae, opportunistic infections and neoplasms.
In addition to CD4+ T cells, the host range of HIV includes cells of the mononuclear phagocytic lineage (Dalgleish, A. G. et al., supra), including blood monocytes, tissue macrophages, Langerhans cells of the skin and dendritic reticulum cells within lymph nodes. HIV is also neurotropic, capable of infecting monocytes and macrophages in the central nervous system causing severe neurologic damage. Macrophage/monocytes are a major reservoir of HIV. They may interact and fuse with CD4-bearing T cells, causing T cell depletion and thus contributing to the pathogenesis of AIDS.
Anti-HIV Drugs
Intensive efforts are currently under way to develop therapies to prevent or intervene in the development of clinical symptoms in HIV-infected individuals. For the most part, efforts have been focused on the use of nucleoside analogue drugs such as AZT (azidothymidine), and on other dideoxynucleoside derivatives such as ddA, ddT, ddI, and ddC. These drugs inhibit the viral enzyme, reverse transcriptase, thereby inhibiting de novo infection of cells. However, once viral infection has been established within a cell, viral replication utilizes host cell enzymes. Thus, drugs which inhibit only reverse transcriptase would be expected to have limited effects. While the spread of free virus within the organism may be blocked, the mechanisms of syncytium formation and pathogenesis through direct intercellular spread remain.
A very small number of HIV-infected T cells can fuse with, and eventually kill, large numbers of uninfected T cells through mechanisms based on viral surface antigen expression. In vitro studies have demonstrated HIV replication even in the continued presence of nucleoside analogues in prolonged culture. Drugs targeting other viral processes are also being developed, such as soluble CD4 and dextran sulfate to inhibit viral binding, alpha interferons and "ampligen" to inhibit viral budding, and castanospermine to inhibit the processing of the viral glycoproteins. These drugs are still in early stages of testing. The actual processes of HIV intracellular replication and protein synthesis have not been specifically targeted because these viral functions were thought to reflect the mere pirating of normal host processes through host mechanisms.
Immunotoxins and Their Limitations
Immunotoxins, have been developed by conjugating a protein toxin to a monoclonal antibody via a linker for targeted therapy, in particular, of tumors (Vitetta, E. S. et al., Ann. Rev. Immunol. 3:197-212 (1985)). In principle, an injected immunotoxin is transported through the blood stream to the targeted tissue, penetrates the tissue, binds to the individual cells expressing the antigen to which the antibody is directed. The toxin bound to the antibody then acts in a highly localized manner to destroy only the cells to which the antibody is bound. All three components of the conjugates are important for the specific achievement of cytotoxicity: the antibody enables the conjugate to be retained in the target tissue by binding to a specific cell-surface antigen, which enhances cellular uptake by the target cells. The linker keeps the toxin bound to the antibody and inactive while in circulation, but allows for rapid release of the active toxin inside the target cells. The toxin kills the cell by inhibiting cellular protein synthesis, or by some other related mechanism.
Some of the most cytotoxic substances known are protein toxins of bacterial and plant origin (Frankel, A. E. et al., Ann. Rev. Med. 37:125-142 (1986)). The cytotoxic action of these molecules involves two events--binding the cell surface and inhibition of cellular protein synthesis. The most commonly used plant toxins are ricin and abrin; the most commonly used bacterial toxins are diphtheria toxin and Pseudomonas exotoxin A.
In ricin and abrin, the binding and toxic functions are contained in two separate protein subunits, the A and B chains. The ricin B chain binds to the cell surface carbohydrates and promotes the uptake of the A chain into the cell. Once inside the cell, the ricin A chain inhibits protein synthesis by inactivating the 60S subunit of the eukaryotic ribosome Endo, Y. et al., J. Biol. Chem. 262: 5908-5912 (1987)).
Diphtheria toxin and Pseudomonas exotoxin A are single chain proteins, and their binding and toxicity functions reside in different domains of the same protein chain. In diphtheria toxin, the C-terminal domain inhibits protein synthesis by ADP-ribosylation of the elongation factor, EF2. The two activities are separate, and the toxin elicits its full activity only after proteolytic cleavage between the two domains. Pseudomonas exotoxin A has the same catalytic activity as diphtheria toxin.
The use of diphtheria toxin-based immunotoxins is limited by the fact that most people have been immunized against diphtheria toxin. The use of ricin-based immunotoxins is also limited because these immunotoxins exhibit specific toxicity only in the presence of lactose, which at high concentrations competes with the cell surface carbohydrates for the B chain binding sites. An alternative approach has been developed to use ricin A chain or "single chain ribosome inactivating protein" (SCRIP) in the preparation of immunotoxins.
Single Chain Ribosome Inactivating Proteins (SCRIPs) and Their Potential Application in Antiviral or Tumor Therapy
Trichosanthin, as well as the novel protein of the present invention, belong to the family of single chain ribosomeinactivating proteins (SCRIPs). SCRIPs are highly active at inactivating ribosomes in cell-free systems, but are relatively nontoxic to intact cells.
SCRIPs, also known as type 1 ribosome-inactivating proteins, catalytically inhibit in vitro eukaryotic protein synthesis (Stripe, F. et al., FEBS Lett. 195:1-8 (1986)). They specifically cleave the N-glycosidic linkage of adenosine at residue A4324 of eukaryotic 28S RNA (Endo, Y. et al., J. Biol. Chem. 262:8128-8130 (1987)). This impairs the interaction of the elongation factor EF2 with the 60S ribosomal subunit, thus abrogating polypeptide chain elongation. SCRIPs are basic proteins with a pI in the range of pH 8 to 10, and molecular weights in generally in the range of about 24 to 33 kDa.
A wide variety of SCRIPs are found in plants. In addition to trichosanthin, other plant-derived SCRIPs include Momordica-derived inhibitors (Barbieri, L. et al., Biochem. J. 186:443-452 (1980); Jimenez, A. et al., Annu. Rev. Microbiol. 39:649-672 (1985)), MAP 30, a protein recently isolated by some of the present inventors from Momordica charantia, which has potent anti-HIV activity with little cytotoxicity (Lee-Huang, S. et al., FEBS Lett. 272:12-18 (1990)), dianthins (Stripe, F. et al., Biochem. J. 195:399-405 (1981)), gelonin (Stripe, F. et al., J. Biol. Chem. 255:6947-53 (1980)), and the pokeweed anti-viral proteins (PAP) (Irvin, J. D., Arch. Biochem. Biophys. 169:522-528 (1975); Irvin, J. D. et al., Arch. Biochem. Biophys.200:418-425 (1980); Barbieri, L. et al., Biochem. J. 203:55-59 (1982)). Many of these SCRIPs are antiviral agents, and some also exhibit selective antitumor activity. SCRIPS and their conjugates, including trichosanthin (McGrath, M. S. et al., Proc. Natl. Acad. Sci. USA 86:2844-2848 (1989)) and PAP-anti-CD4 conjugate (Zarling, J. M. et al., Nature 347:92-95 (1990)) have been reported to possess anti-HIV activity, but have variable degrees of nonspecific cytotoxicity.
Recently, the primary amino acid sequence of .alpha.-trichosanthin and molecular models for abrin A-chain and .alpha.-trichosanthin were reported (Collins, E. J. et al., J. Biol. Chem. 265:8665-8669 (1990)). This .alpha.-trichosanthin sequence differs substantially from sequences reported by others for trichosanthin (Zhang, X. et al., Nature 321:477-478 (1986); Maraganore, J. Met al., J. Biol. Chem. 262:11628-11633 (1987)). This proposed structural model of trichosanthin differs from a previous model generated from x-ray diffraction data (Pan, K. St al., Sci. Sin. Ser B. (Chem. Biol. Agric. Med. & Earth Sci.) 30:386-395 (1987)). All of these trichosanthins were isolated from root tubers of the same species of T. kirilowii. However, no systematic comparison of the cytotoxicity and bioactivities of these proteins have been reported. In view of the therapeutic potential of these proteins, studies on the relationship between their structure and function may lead to the rational design and targeted development of safer and more effective second generation anti-HIV drugs.
M. S. McGrath et al. (supra) reported that GLQ 223, a SCRIP isolated from T. kirilowii, selectively inhibits HIV replication. This compound demonstrated dose-dependent anti-HIV activity in both acutely and chronically infected T cells and monocytes/macrophages. This discovery led to the rapid clinical testing of GLQ 223 as an anti-AIDS therapeutic. Treatment of cells with GLQ 223 resulted in selective inhibition of the synthesis of viral DNA, RNA, and protein, with less effect on cellular synthesis. Inhibition of viral replication occurred at GLQ 223 concentrations that had less of an effect on uninfected cells.
The mechanisms of the selective anti-HIV activity of GLQ 223 is not known. It has not been established whether this activity is associated with the ribosome-inactivating or the abortifacient activity of this compound. Two possible mechanisms are immediately apparent. Selective binding or uptake of GLQ 223 by infected cells could be responsible for its selective action on infected cells. Once inside the infected cells, the compound could act non-specifically, via its ribosome inactivating function. Alternatively, selectivity of the agent may arise from differential effects on viral versus cellular components, resulting in greater inhibition of viral compared to cellular nucleic acid and protein synthesis.
Lifson et al., U.S. Pat. No. 4,795,739 (issued Jan. 3, 1989), discloses that plant proteins, including trichosanthin, reduce viral antigen expression in HIV-infected cells, and are selectively toxic to HIV-infected cells. These proteins are said to be useful for treating HIV infections in humans.
The cytotoxic side effects of trichosanthin are well known (Qian, R. Q. et al., Acta Chemica Sinica 39:927-931 (1981); Gu, Z. et al. Acta Chemica Sinica 43:943-945 (1984)) since it has been used for centuries in Chinese traditional medicine, in particular for abortion and treatment of trophoblastic tumors (Li, S.C. (1596) Pen Ts'ao Kang Mu, (Chinese Pharmaceutical Compendium) reprinted by People's Medical Publishing House, Beijing (1977); Cheng, K. F., Obstet. Gynecol. 59:494-498 (1982); Chan, W. Y. et al., Contraception 29:91-100 (1984)). The cytotoxicity of GLQ 223 has also been documented in recent clinical trials in the United States (Palcca, J., Science 247:1406 (1990)). Thus, concerns with therapeutic safety of trichosanthin (Palcca, supra) and other plant derived agents, represent serious obstacles in their utility as anti-HIV therapeutics.